Recombinant plasmid and a method of controlling the effects of Yersinia pestis

ABSTRACT

Described is a plasmid prepared by recombinant techniques which is used to prepare a vaccine against  Y. pestis.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.08/302,423, filed Sep. 8, 1994, now U.S. Pat. No. 6,638,510 B1.

LICENSING RIGHTS

The United States government may have licensing rights to thisapplication in accordance with U.S. Public Health Service Grant AI19353.

TECHNICAL FIELD

The present invention is concerned with bubonic plague caused byYersinia pestis and vaccines for treating same.

BACKGROUND ART

Experimental plague in mice, caused by Yersinia pestis, is mediated bytwo distinct types of virulence factors. Members of the first categoryserve as whole animal or tissue invasins by promoting dissemination ofthe organisms into visceral organs following infection by peripheralroutes of injection (e.g. intraperitoneal or subcutaneous). Mutantslacking one or more tissue invasins can exhibit significant reduction invirulent (50% lethal dose>10² to 10⁷ bacteria) by peripheraladministration but retain essentially full lethality (50% lethal doseca. 10² bacteria) upon intravenous injection (4). Examples of this group(6, 16, 48) include the outer membrane (46) plasminogen activator (1)mediated by a ca. 10 kb pesticin or Pst plasmid (12, 41) and a series ofiron repressible outer membrane peptides (10, 40) encoded by adelectable ca. 100 kb chromosomal segment (11, 22).

Examples of the second category function to promote lethality followinginfection by the intravenous route, known to facilitate immediatetransport of the bacteria to favored niches within visceral organs (4).Mutational loss of these lethal factors causes qualitative (intravenous50% lethal dose>10⁷ bacteria) decreases in virulence. Included in thisgroup are certain ca. 70 kb low calcium response or Lcr plasmid encodedproteins: V antigen (9, 27), others termed Yops (18, 19, 33, 47): YopE(23, 35, 43, 44, 47), YopH (3, 34, 44), and probably YpkA (13), as wellas chromosomally encoded antigen 4 or pH 6 antigen (20) and possibly themurine exotoxin encoded by the ca. 100 kb Tox plasmid (32.).Considerable effort has been spent in study of the regulation,processing, and delivery of these proteins to host cells (2, 15, 26, 28,30, 31, 35).

The effectiveness of the immune response directed against members of thesecond category of virulence factors has only been reported for Vantigen. Some (17, 24, 27, 39, 49, 50) but not all (5) antibodiesdirected against this 37 kDa exported (8, 17, 43, 44) protein providedsignificant passive protection against experimental plague in mice. Thiseffect was associated with release of a potent immunosuppressive blockpreventing both synthesis of cytokines (27) and formation of protectivegranulomas (50).

Plague vaccines have been identified in U.S. Pat. No. 3,137,629. Thepatent describes a process for producing killed plague vaccines whichimmunizes mice and guinea pigs by growing Pasteurella pestis, killingthe strain through mechanical action and solubilizing the extract instrong alkaline solution, and then preparing parenteral vaccine byreducing the pH value of the soluble P. pestis antigenic solution to aneutral pH.

It is an object of the present invention to prepare a plasmid byrecombinant techniques.

It is another object of the present invention to prepare an antigenencoded by the recombinant plasmid.

It is a further object of the present invention to control the effect Y.pestis has on mammals by utilizing a vaccine to Y. pestis constitutingthe antigen noted above.

SUMMARY OF THE INVENTION

The present invention is concerned with a plasmid prepared byrecombinant techniques having the construct shown in FIG. 1.

Also described is a protein encoded by the plasmid shown in FIG. 1,capable of inducing a protective antibody response.

The invention is further concerned with a method of controlling theeffects of Y. pestis in mammals comprising the steps of:

a) providing a vaccine comprised of the protein encoded by the constructof FIG. 1; and

b) treating a mammal in need thereof with an effective anti-Y. pestisamount of the vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A is a scheme of construction of a recombinant plasmid formed byjoining the DNA encoding the signal sequence and IgG binding domains ofstaphylococcal protein A and that of all but the 201 N-terminal basepairs of Y. pestis V antigen; antibody to which is capable of effectingimmunological treatment against Y. pestis;

FIG. 1B cites a restriction endonuclease attack on the V antigen;

FIG. 2 is a silver-stained extended SDS gel of whole cells of E. coli BL21 containing the vector plasmid pKK223-3 (lane 1) or recombinantplasmid pKVE14 (lane 2);

FIG. 3 are immunoblots prepared with rabbit polyclonal anti-V antigen(A) mouse monoclonal anti-V antigen 15A4.8 (B) and mouse monoclonalanti-V antigen 3A4.1 (C) directed against whole cells of E. colicontaining the vector plasmid pKK223-3 (lane 1) or recombinant plasmidpKVE14 (lane 2);

FIG. 4 are immunoblots prepared with rabbit anti-native V antigenpurified from Y. pestis KIM (A), anti-recombinant V antigen (B),anti-protein A-V antigen fusion protein (C), and anti-Protein A (D)directed against Ca²⁺-starved whole Lcr⁻ cells of Y. pestis KIM (lane1), Lcr⁺ cells of Y. pestis KIM (lane 2), Lcr⁻ cells of Y.pseudotuberculosis PB1 (lane 3), Lcr⁺ cells of Y. pseudotuberculosis PB1(lane 4), Lcr⁻ cells of Y. enterocolitica WA (lane 5), and Lcr⁺ cells ofY. enterocolitica WA (lane 6);

FIG. 5 is a chart of antiserum for passive immunization against native Vantigen, recombinant V antigen, recombinant protein A-V antigen fusionand recombinant protein A;

FIG. 6 are immunoblots prepared with rabbit anti-native V antigen (A) ormouse monoclonal 17A5.1 anti-V antigen (B) directed against truncatedprotein A (PA) (lane 1), protein A-V antigen fusion peptide (PAV) (lane2), PAV partially hydrolyzed by formic acid (lane 3), PAV partiallyhydrolyzed by formic acid and passed through the IgG Sepharose 6FFcolumn (lane 4), whole Lcr⁺ cells of Yersinia pestis KIM (lane 5), andwhole LCR^(−·) cells of Y. pestis KIM (lane 6); A-V_(d), V_(o), V_(d),and A indicate the positions of PAV, native V antigen (37 Kda),truncated V antigen (29.5 kDa), and truncated Protein A, respectively.Human γ-globulin was used to block nonspecific reactions of monoclonalantibodies against IgG-binding domains of Protein A (26); and

FIG. 7 are immunoblots prepared with rabbit anti-protein A-V antigenfusion peptide (A) and anti-truncated protein A (B) directed againstwhole cells of Escherichia coli containing the vector plasmid pKK223-3(lane 1) or recombinant plasmid pKVE14 (lane 2); Also shown arereactions against disrupted and centrifuged whole cells of E. coli(wpKE14) (lane 3) and further fractionation of V antigen bychromatography on phehyl-Sepharose CL-4B (lane 4), DEAE(diethylaminothyl) cellulose (lane 5), Sephacryl S-300SF (lane 6),calcium hydroxylapatite (lane 7), and a second passage on DEAE cellulose(lane 8).

DESCRIPTION OF THE BEST MODE

The medically significant yersiniae (Yersinia pestis, Y.pseudotuberculosis, and Y. enterocolitica) are known to share a ca. 70kb low calcium response (Lcr) plasmid that mediates restriction ofvegetative growth at 37° C. in Ca²⁺-deficient media while promotingselective synthesis of virulence factors including V antigen. Thelatter, encoded by lcrV on the Lcr plasmid, is established as a 37 kDaprotective antigen capable of undergoing possible autoproteolytichydrolysis. In this study, lcrV of Y. pestis was cloned under control ofthe strong tac promoter into protease-deficient Escherichia coli BL21.The resulting recombinant V antigen, like native V antigen, underwentdegradation during purification yielding major peptides of ca. 36,35,34and 32 to 29 kDa. Rabbit γ-globulin raised against this mixture ofcleavage products provided partial but significant protection against 10minimal lethal doses (MLD) of the three species. To stabilize V antigenand facilitate its purification, plasmid pPAV13 was constructed so as toencode a fusion of lcrV and the structural gene for staphylococcalprotein A (e.g. all but the first 67 N-terminal amino acids of V antigenand the signal sequence plus IgG binding domains but not cell-wallassociated region of protein A). The resulting protein A-V antigenfusion peptide (PAV) could be purified to homogeneity in one step IgGaffinity chromatography and was found to be stable thereafter. Rabbitpolyclonal γ-globulin directed against PAV provided substantial passiveimmunity against 10 MLD of Y. pestis and Y. pseudotuberculosis but wasineffective against Y. enterocolitica.

The vaccine as described herein is generally applied by parenteraladministration to mammals in need thereof.

The vaccine of the present invention is generally administered in theform of pharmaceutical compositions comprising a pharmaceuticallyacceptable vehicle or diluent. Such compositions are generallyformulated in a conventional manner utilizing liquid vehicles ordiluents as appropriate to the mode of desired administration: forparenteral administration (e.g. intramuscular, intravenous,intradermal), in the form of injectable solutions or suspensions, andthe like. For use as a vaccine in a mammal, including man, it is givenin an amount of about 0.5-100 mg/kg.

The vaccine described herein is used in conjunction with normalpharmaceutical excipients to facilitate storage and use.

The present invention is further illustrated by the following examples.These examples are provided to aid in understanding of the invention andnot to be construed as a limitation thereof.

Materials and Methods

Bacteria. Escherichia coli K-12 XL1-Blue {recA1 enda1 gyrA96 thi-1hsdR17 supE44 relA1 lac [F′ proAB lacI^(q)ZΔM15Tn10 (tet^(r))]}(Stratagene, La Jolla, Calif.) was used as a host for geneticengineering manipulations. Protease-deficient E. coli BL21 {F⁻ ompT lonr_(B)- m_(B)-} (Novagen, Madison, Wis.) was used for expression ofcloned genes (17). Mice passively immunized with the products of clonedgenes were challenged with wild type cells of Y. enterocolitica WA (10)or Y. pseudotuberculosis PB1/+ (9). This purpose was accomplished withY. pestis KIM by use of a nonpigmented mutant (20,59) known to lack aspontaneously deletable ca. 100 kb chromosomal fragment encodingfunctions of iron transport and storage (14,28); the isolate in questionretained all other known chromosomally encoded virulence functions aswell as the Tox, Lcr, and Pst plasmids (13,57). Nonpigmented mutants ofthis phenotypic background are virulent in mice by the intravenous (50%lethal dose ca. 10 bacteria, 61) but not by peripheral routes ofinfection (50% lethal dose>10⁷ bacteria, 21).

Plasmids. Salient features of plasmids used in this study are shown inTable 1. The vector pKK223-2 containing the tac promoter (Pharmacia,Uppsala, Sweden) was used to express a portion of the lcrGVH-yopBDoperon of Y. pestis 358 (22) as described below. The vector pRIT5(Pharmacia) encoding the sequence of Protein A of Staphylococcus aureuswas used for preparation of gene fusions. The recombinant plasmid pBVP5containing the lcrGVH-YopBD operon of Y. pseudotuberculosis (38) servedas the source of lcrV in preparing this construction.

TABLE 1 Characterization of Deletional Variants of HindIII Fragment FromThe IcrGVH-yopBD Operon of Yersinia pseudotuberculosis 995. DesignationEncoding Size Of Of Size of V Plasmid Fragment Operon V Antigen Antigen(kDa) pBVP5 ˜3,500 1crGVHyopBD V₀ 37.3 pBVP513D 2,184 1crGVH V₀ 37.3pBVP53D 1,484 1crGV₁ V₁ 31.5 pBVP514D 1,160 1crGV₂ V₂ 19.3 pBVP515D 8781crGV₃ V₃  8.5 pBVP58D 705 1crGV₄ V₄  2.0 pBVP55D 546 1crG₁ — —

Molecular weights of truncated V antigens were calculated from thedeletion terminus as determined by nucleotide sequencing; actual valuesmay be slightly greater due to translational overruns into the vectorpolylinker region.

DNA Methods. The preparation of plasmid DNA, digestion with restrictionenzymes, ligation, and transformation of E. coli were undertakenessentially as described by Maniatis et al (29). The 3.5 kb HindIIIfragment of the Lcr plasmid of Y. pestis 358 (22,38) was introduced intoexpression vector pKK223-3. The resulting recombinant plasmid pKVE14 wasthen selected where the direction of transcription of the lcrGVHsequence corresponds to the direction of action of the tac promoter.

The schema used to construct pPAV13 containing a hybrid gene encoding aportion of Protein A of S. aureus and lcrV of Y. pseudotuberculosis isshown in FIG. 1A. The 1.5 kb EcoRV fragment of recombinant plasmid pBVP5(38) was introduced into the vector pRIT5 encoding truncated Protein A(PA). The latter, either alone or fused with V antigen, maintained itssignal sequence and most IgG-binding domains but lost the regionmediating association with the bacterial cell surface (41,42) (FIG. 1B).PA does not contain cysteine and is thus unable to form disulfidebridges between itself and a hybrid domain (60). As a consequence ofthis fusion, lcrV lost 201 bp which thus deleted the first 67 aminoacids comprising the N-terminal portion of V antigen. The resultingProtein A-V antigen fusion peptide (PAV) thus contained 305 N-terminalamino acids from Protein A and 259 C-terminal amino acids from V antigen(FIG. 1B).

Purification of Recombinant V Antigen. Cells of E. coli BL21 (pKVE14)were grown in fermenters as described previously (5) in mediumcontaining 3% Sheffield NZ Amine, Type A (a pancreatic hydrolysate ofcasein which contains mixed amino acids and peptides and is used tofacilitate the growth of bacteria) (Kraft, Inc., Memphis, Tenn.), 0.5%NaCl, 1% lactose, and ampicillin (100 μg/ml) at 37° C. and harvested bycentrifugation (10,000×g for 15 min) at an optical density (620 nm) ofabout 1.2. After disruption in a French pressure cell (SLM Instruments,Inc., Urbana, Ill.) and removal of insoluble matter by centrifugation(10,000×g for 30 min), V antigen was subjected to purification by anestablished procedure (5). The method involved use of hydrophobicinteraction chromatography with phenyl-Sepharose CL-4B (Pharmacia), ionexchange chromatography with DEAE cellulose (Whatman Inc., Clifton,J.J.), gel filtration chromatography with Sephacryl S-300SF (trademarkof Pharmacia Biotechnology Group for acrylic resin for chromatograhicseparation of proteins), and Bio-Gel HTP (trademark of Bio-Rad,Richmond, Calif., for calcium hydroxyapatite chromatography). Theoriginal procedure was supplemented by a second chromatographicseparation of DEAE cellulose (linear gradient from 0.35 M NaCl) in orderto remove high molecular weight material peculiar to E. coli.

Preparation of PA and PAV. Cells of E. coli transformed with pPAV13 orPRIT5 were grown to late log phase at 37° C. in Luria broth containingampicillin (50 μ/ml). Purification of these recombinant proteins wasaccomplished by affinity chromatography on IgG Sepharose 6FF (Pharmacia)according to directions supplied by the manufacturer. Briefly, theprocedure involved harvesting the organisms by centrifugation (10,000×gfor 15 min) with resuspension at a ca. 10-fold increase in number in0.01 M Tris.HCl, pH 8.0 (column buffer). Lysis was accomplished byaddition of lysozyme (5 mg/ml) and, after incubation for 1 h, furtheraddition of Triton X-100 (trademark of Rohm & Haas Co. for a nonionicdetergent comprised of octyl phenoxy polyethoxy ethanol having an HLB:13.5_(j) (0.1%) whereupon incubation was continued for 3 to 4 h. Afterclarification by centrifugation (10,000×g for 30 min), samples of 400 mlof the resulting enriched periplasm were passed through a column (10×100mm) containing a 10 ml packed volume of affinity resin that selectivelybound PA or PAV. After addition and elution of 10 void volumes of columnbuffer to remove contaminating matter, the recombinant proteins wereeluted with 0.2 M acetic acid (ca. pH 3.4), immediately frozen, and thenlyophilized. Resulting purified PA and PAV were then used directly forqualitative analysis and immunization.

Acid Hydrolysis of PAV. Purified PAV was treated with 70% formic acidfor 20 h at 30° C. to cleave the four labile Asp-Pro peptide bondswithin the Protein A domain (60) and the additional site located at thejunction with V antigen (41) (FIG. 1B). After dialysis against columnbuffer, the partial hydrolysate was again passed through the IgGSepharose 6FF column as described above. In this case, the V antigenmoiety plus fragments of PA lacking IgG binding sites were immediatelyeluted whereas residual unhydrolyzed PAV remained bound to the affinityresin.

Antisera. The same lot of refined rabbit polyclonal anti-V antigencharacterized previously (40) was used as a positive immunologicalcontrol. Monoclonal antibodies directed against V antigen have beendefined (3). These reagents consisted of two groups: monoclonals 3A4.1,17A5.1. and 17A4.6 that reacted with nonconformational epitopes locatedwithin the last 50 amino acids comprising the C-terminal part of Vantigen (amino acids 276 to 326) and monoclonal 15A4.8 that reacted withan internal nonconformational epitope located between amino acids 168 to275 (37).

Rabbit polyclonal antisera was raised against PA and PAV with Freund'sadjuvant as described previously (62). TiterMax™ adjuvant (Hunter'sTiterMax #R-1, CytRx Corp., Norcross, Ga.) was used to immunize rabbitsagainst recombinant V antigen plus its degradation products purifiedfrom E. coli BL21 (pKVE14). Antisera prepared against recombinant Vantigen or fusion proteins were not absorbed with material from Lcr⁻bacteria although highly purified γ-globulin was isolated from thesereagents as described previously (62). Antisera raised against V antigenpurified from Y. pestis or E. coli BL21(pKVE14) is termed anti-native Vantigen or anti-recombinant V antigen, respectively.

Immunoblotting. Alkaline phosphatase conjugated with anti-rabbit oranti-mouse IgG (Sigma Chemical Co., St. Louis, Mo.) were used assecondary antibodies in immunoblotting by procedures essentiallyidentical to those already defined (51,52). In order to preventnonspecific reactions of antibodies with PA and PAV, the nitrocellulosefilter was first blocked with 5% fetal calf serum as usual and thenincubated overnight in a solution of 1% normal human γ-globulin(Calbiochem, San Diego, Calif.). Human γ-globulin (0.5%) was also addedto solutions of primary and secondary antibodies (26). In addition,Fc-specific anti-mouse IgG (A-1418, Sigma) was used as a secondaryantibody during immunoblotting of fusion proteins and their derivativeswith monoclonal antibodies.

Passive Immunity. The ability of highly purified γ-globulin obtainedfrom unabsorbed rabbit polyclonal antisera raised against recombinant Vantigen, PA, and PAV to provide passive immunity was assayed by definedmethods (40,62). Briefly, this procedure involved intravenous injectionof 10 minimum lethal doses (MLD) of Y. pestis (10² bacteria), Y.pseudotuberculosis (10² bacteria), or Y. enterocolitica (10³ bacteria)followed by intravenous administration of either 100 μg or 500 μg ofpurified γ-globulins on postinfection days 1, 3, and 5.

Miscellaneous. Peptides were located in sodium dodecylsulfate-polyacrylamide gel electrophoresis gels, prepared as definedpreviously (51,52), by silver staining (36). Soluble protein wasdetermined by the method of Lowry et al. (27).

Results

Degradation of Recombinant V Antigen. Recombinant plasmid pKVE14containing the lcrGVH-ypoBD operon of Y. pestis under control of the tacpromoter was transferred into protease-deficient E. coli BL21. Aftergrowth in fermenters, the bacteria were disrupted and the resultingextract was used to prepare nearly homogenous recombinant V antigenusing a method established for Ca²⁺-starved cells of Y. pestis (5). Anadditional step involving a second separation with DEAE cellulose wasnecessary to eliminate major higher molecular weight proteins present inE. coli cytoplasm.

The initial specific activity of recombinant V antigen was almost 5-foldgreater than that obtained from Y. pestis starved for Ca²⁺ (5).Nevertheless, significant loss of precipitin activity occurred duringevery step of purification (Table 2). This phenomenon, as judged by asilver-stained extended lane gel (FIG. 2), reflected gradual loss of thenative 37 kDa form with emergence of ca. 36 kDa, 32 kDa, and possiblysmaller peptides. Analysis by immunoblotting was undertaken to provethat these new peptides shared epitopes with and thus arose from nativeV antigen. Use of rabbit polyclonal anti-native V Antigen (FIG. 3A) ormouse monoclonal antibody 15A4.8, directed against a centrally locatedepitope (FIG. 3B), demonstrated emergence of ca. 36, 35, and 34 kDadegradation products early during the course of purification with laterappearance of a series of smaller fragments ranging from 32 to 29 kDa.The latter were not recognized by mouse monoclonal antibody 3A4.1directed against an epitope located near the C-terminal end (FIG. 3C).These findings indicate that recombinant V antigen produced inprotease-deficient E. coli BL21 undergoes evident spontaneousdegradation in a manner similar to that observed from native V antigenexpressed in Y. pestis (5). Furthermore, patterns observed uponimmunoblotting with monoclonal antibodies indicate that the C-terminalportion of V is involved in this process.

TABLE 2 Purification Of Recombinant V Antigen From A Cell-Free ExtractOf Escherichia coli BL21 (pKVE14) Total Protein Protein V Antigen TotalSpecific Preparation Vol. (ml) (mg/ml) (mg) (U/ml) V-antigen (U)Activity % Recovery Crude Extract 200 26 5,200 280 56,000 11 (100)Phenyl-Sepharose 220 1.6 350 140 30,800 88  55 CL-4B DEAE 40 1.5 60 1706,800 113   12.1 Cellulose 24 0.7 17 140 3,360 200    6.0 SephacrylS300SF Ca Hydroxylapatite 35 0.25 8.8 50 1,750 200    3.1 DEAE Cellulose18 0.1 1.8 15 270 150    0.5

The unit of V antigen was defined as the reciprocal of the highestdilution capable of forming a visible precipitate against a standardizedlot of rabbit polyclonal monospecific antiserum by diffusion in agarunder conditions described previously (Lawton et al, 1963; Brubaker etal., 1987).

Passive Immunity Mediated by Anti-recombinant V Antigen. A portion ofthe purified lot of recombinant V antigen described above was used toimmunize rabbits. Immunoblots of the resulting unabsorbed antisera (FIG.4B) and control absorbed anti-native V antigen (FIG. 4A) versusCa²⁺-starved whole yersiniae were identical indicating that the reagentwas monospecific. Both antisera were tested for ability to conferpassive immunity against intravenous infection with yersiniae. As shownin FIG. 5, the control anti-native V antigen provided complete, partial,and insignificant protection against Y. pestis, Y. pseudotuberculosis,and Y. enterocolitica, respectively. Antirecombinant V antigen promoteda similar degree of passive immunity except that that directed againstY. pestis was not absolute.

Characterization of PA and PAV. Additional constructions encodingtruncated staphylococcal Protein A either alone or fused with V antigen(FIG. 1) were found, after transformation into E. coli BL21, to promotesignificant synthesis of PA and PAV, respectively as judged by intensityof the specific immunoblots described below. PA and PAV were purified inone step with IgG Sepharose 6FF and then analyzed by immunoblotting.Anti-native V antigen reacted nonspecifically with PA (FIG. 6A, lane 1)and both specifically and nonspecifically with PAV (FIG. 6A, lane 2).Proof that the salient peptides shown in lanes 2, 3, and 4 of FIG. 6Areacted specifically rather than nonspecifically with anti-native Vantigen was obtained by blocking PA with human γ-globulin and thenimmunoblotting with monoclonal anti-V antigen. This process preventedvisualization of PA (FIG. 6B, lane 1) thus demonstrating that theremaining detectable bands represent a specific interaction with anepitope of V antigen. Multiple bands appearing in samples of both PA andPAV (FIG. 6A, lanes 1, 2) reflect accumulation in the periplasm of E.coli BL21 of the synthesized PA domain in both native and degraded formsas described by others (16). To prove that the V antigen domain of thefusion protein was stable, a sample of purified PAV was hydrolyzed with70% formic acid to cleave acid labile Asp-Pro sites defined in FIG. 1B,neutralized, and then applied to the affinity column. Essentially puretruncated V antigen (V_(d)) emerged immediately (FIG. 6, lane 4); theabsence of multiple bands in this sample provides evidence for thestability of V antigen within PAV.

Stability of PAV. The number of total units of near-homogenous PAVrecovered after chromatography on IgG Sepharose 6FF was alwaysessentially identical to that present in the crude extract applied tothe affinity column. No significant loss of purified PAV occurred duringstorage in 0.01 M Tris. HCl, pH 7.8 for 1 week at 4° C.

Passive Immunity Mediated by Anti-PAV. Preparations of homogenousγ-globulin were isolated from unabsorbed rabbit antisera raised againstPA and PAV purified by affinity chromatography. Control anti-V antigen(FIGS. 4A and 4B) and anti-PAV (FIG. 4C), but not anti-PA (FIG. 4D),reacted with V antigens of all three Yersinia sp. High molecular weightantigens (ca. 70 kDa) common to both Lcr⁺ and Lcr⁻ yersiniae wererecognized by both anti-PAV (FIG. 4C) and anti-PA (FIG. 4D). Anti-PAV(FIG. 7A) but not anti-PA (FIG. 7B) also identified the same degradationproducts of V antigen that were detected by the antisera characterizedpreviously (FIG. 3). These findings verify that anti-PAV containsantibodies directed against epitopes unique to V antigen and providefurther evidence that its degradation occurs at the C-terminal end.

Anti-PA and anti-PAV were tested for ability to confer passive immunityagainst intravenous infection with yersiniae. As shown in FIG. 5,anti-PA failed to provide protection whereas anti-PAV was highlyeffective against Y. pestis and Y. pseudotuberculosis but not Y.enterocolitica.

Discussion

Since its discovery, V antigen has been ascribed a role as protectiveantigen in conferring immunity to plague (8). Experimental evidencesupporting this assumption was initially limited to the findings thatactive immunization with V antigen-rich fractions or passiveimmunization with antisera raised against such fractions providedprotection against disease (23). The possibility thus remained that oneor more additional antigens contributed to the immune state described byearly workers. This concern was minimized upon introduction ofpreparative methods that permitted recovery of nearly homogenous Vantigen (5). Nevertheless, antisera raised against lots purified by useof these procedures often contained antibodies directed against highlyantigenic contaminating proteins, especially Yops, present at tracelevels in the final product used for immunization. Although theseantibodies were readily removed by absorption with cross-reactingmaterial, this process necessitated introduction of bacterialmacromolecules including lipopolysaccharide that might stimulatenonspecific resistance to infection. To minimize this possibility, itbecame necessary to purify the γ-globulin from absorbed antisera(40,62).

Concerns that these precautions, undertaken to assure monospecificity ofanti-V antigen, had inadvertently introduced uncontrolled variables werelargely eliminated by use of γ-globulin purified from antisera raisedagainst highly purified V antigen cloned in E. coli. However, thisprocess was also unsatisfactory due to the occurrence of markeddegradation throughout the course of purification. As a result, only afraction of the final product consisted of the 37 kDa primary lcrVproduct. Although γ-globulin purified from unabsorbed antiserum raisedagainst this mixture provided satisfactory passive immunity, yields ofantigenic material were insufficient to permit widespread immunization.The observation that cloned V antigen expressed in theprotease-deficient background of E. coli BL21, like native V antigenpurified from Y. pestis, underwent marked degradation duringpurification further suggests but does not prove that this process isautocatalytic.

Problems concerning specificity and degradation were resolved upondevelopment of the fusion protein PAV that could be isolated at highyield as a homogenous stable protein in a single step. Antisera raisedagainst PAV were somewhat more effective in providing protection againstY. pestis, and especially Y. pseudotuberculosis than wasanti-recombinant V antigen. This finding emphasizes that passiveimmunity mediated by anti-V antigen does not require interaction withN-terminal epitopes because the latter were absent in PAV. Theindependent observation that the N-terminal end of V antigen was poorlyantigenic (37) is consistent with this conclusion.

Detailed information with respect to the drawing figures is as follows.

FIG. 1. Scheme of construction of recombinant plasmid of pPAV13 encodingstaphylococcal protein A-V antigen fusion protein (PAV) (A) andcharacterization of PAV (B). Sites of restriction endonuclease attackare designated; Ap and Cm are locations of markers of resistance forampicillin and chloramphenicol, respectively. Lac designates theposition of lacZ which provides selection of recombinant plasmids in thevector pBluescript SK+. The genes lcrG, lcrv, and lcrH comprise aportion of the lcrGVH-yopBD operon of Yersinia pseudotuberculosis 995(38) and the designation Protein A is the truncated Protein A gene ofStaphylococcus aureus. The dark arrows in A represent the hybrid geneencoding PAV shown in B to consist of the signal sequence (S),IgG-binding domains (E to B), the defective domain C′ that has lost theability to bind IgG, and truncated V antigen that has lost the first 67amino acids of its N-terminal portion. Molecular weights in Kilodaltonsare designated for each peptide arising after hydrolysis of theacid-labile Asp-Pro cleavage sites marked by arrowheads (60).

FIG. 2. Silver-stained 12.5% extended sodium dodecylsulfate-polyacrylamide electrophoresis gel of whole cells of Escherichiacoli BL21 containing the vector plasmid pKK223-3 (lane 1) or recombinantplasmid pKVE14 (lane 2). Whole cells of E. coli (pKVE14) were disruptedand centrifuged to prepare a cell-free extract (lane 3) that wasfractionated by chromatography on phenyl-Sepharose CL-4B (lane 4), DEAEcellulose (lane 5), Sephacryl S-300SF (lane 6), calcium hydroxylapatite(lane 7), and a second passage on DEAE cellulose (lane 8). Note thepresence of V antigen in lanes 2 through 8 as a major peptide of 37 kDa.

FIG. 3. Immunoblots prepared with rabbit polyclonal anti-V antigen (A),mouse monoclonal anti-V antigen 15A4.8 (B), and mouse monoclonal anti-Vantigen 3A4.1 (C) directed against whole cells of Escherichia colicontaining the vector plasmid pKK223-3 (lane 1) or recombinant plasmidpKVE14 (lane 2). Also shown are reactions against disrupted andcentrifuged whole cells of E. coli (pKVE14) (lane 3) and furtherfractionation of V antigen by chromatography on phenyl-Sepharose CL-4B(Lane 4), DEAE cellulose (lane 5), Sephacryl S-300SF (lane 6), calciumhydroxylapatite (lane 7) and a second passage on DEAE cellulose (lane8).

FIG. 4. Immunoblots prepared with rabbit anti-native V antigen purifiedfrom Y. pestis KIM (A), anti-recombinant V antigen (B), anti-protein A-Vantigen fusion protein (C), and anti-Protein A (D) directed againstCa²⁺-starved whole Lcr⁻ cells of Y. pestis KIM (lane 1), Lcr⁺ cells ofY. pestis KIM (lane 2), Lcr⁻ cells of Y. pseudotuberculosis PB1 (lane3), Lcr⁺ cells of Y. pseudotuberculosis PB1 (lane 4), Lcr⁻ cells of Y.enterocolitica WA (lane 5), and Lcr⁺ cells of Y. enterocolitica WA (lane6).

FIG. 5. Ability of 0.033 M potassium phosphate buffer, pH 7.0 (None) orcontrol antiserum raised against native V antigen and experimentalantisera raised against recombinant V antigen, protein A-V antigenfusion peptide, and recombinant protein A to provide passive protectionin mice against 10 minimum lethal doses of Lcr⁺ cells of Yersinia pestisKIM, Yersinia pseudotuberculosis PB1, and Yersinia enterocolitica WA.Mice were challenged intravenously and γ-globulins were thenadministered intravenously on postinfection days 1, 3, and 5 at, unlessindicated otherwise, a dose of 100 μg. Lengths of solid bars showsurvival in days of individual mice eventually succumbing to infection;open bars represent survival of independent infected mice until theexperiment was terminated at 21 days. Numbers adjacent to solid barsshow mean survival time in days.

FIG. 6. Immunoblots prepared with rabbit anti-native V antigen (A) ormouse monoclonal 17A5.1 anti-V antigen (B) directed against truncatedprotein A (PA) (lane 1), protein A-V antigen fusion peptide (PAV) (lane2), PAV partially hydrolyzed by formic acid (lane 3), PAV partiallyhydrolyzed by formic acid and passed through the IgG Sepharose 6FFcolumn (lane 4), whole Lcr⁺ cells of Yersinia pestis KIM (lane 5), andwhole Lcr⁻ cells of Y. pestis KIM (lane 6); A-V_(d), V_(o), V_(d), and Aindicate the positions of PAV, native V antigen (37 kDa), truncated Vantigen (29.5 kDa), and truncated Protein A, respectively. Humanγ-globulin was used to block nonspecific reactions of monoclonalantibodies against IgG binding domains of Protein A (26).

FIG. 7. Immunoblots prepared with rabbit anti-protein A-V antigen fusionpeptide (A) and anti-truncated protein A (B) directed against wholecells of Escherichia coli containing the vector plasmid pKK223-3(lane 1) or recombinant plasmid pKVE14 (lane 2). Also shown arereactions against disrupted and centrifuged whole cells of E. coli(pKVE14) (lane 3) and further fractionation of V antigen bychromatography on phenyl-Sepharose CL-4B (lane 4), DEAE cellulose (lane5), Sephacryl S-300SF (lane 6), calcium hydroxylapatite (lane 7), and asecond passage on DEAE cellulose (lane 8).

While the forms of the invention herein disclosed constitute presentlypreferred embodiments, many others are possible. It is not intendedherein to mention all of the possible equivalent forms or ramificationsof the invention. It is understood that the terms used herein are merelydescriptive rather than limiting, and that various changes may be madewithout departing from the spirit or scope of the invention.

References

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1. An isolated and purified recombinant V antigen encoded by the plasmidconstruct pPAV13 as shown in FIG.
 1. 2. An isolated and purifiedrecombinant V antigen lacking the first 67 N-terminal amino acids ofnative V antigen.
 3. The isolated and purified recombinant V antigen ofclaim 1, wherein the plasmid comprises low calcium response regions Vand H and the structural gene for staphylococcal protein A.